Test composition and method for detecting reducing sugars in aqueous fluids

ABSTRACT

TEST COMPOSITION AND METHOD FOR DETECTING REDUCING SUGARS IN AQUEOUS BIOCHEMICAL AND INDUSTRIAL FLUIDS COMPRISING A DRY SOLID MIXTURE OF AN ACID MATERIAL, AN ALKALINE MATERIAL, A CUPRIC SALT AND A SURFACE ACTIVE AGENT CAPABLE OF FORMING A SUBSTANTIALLY MONMOMOLECULAR FILM AT THE LIQUID-VAPOR INTERPHASE OF THE FLUID BEING TESTED.

United States Patent U.S. Cl. 252-408 8 Claims ABSTRACT OF THEDISCLOSURE Test composition and method for detecting reducing sugars inaqueous biochemical and industrial fluids comprising a dry solid mixtureof an acid material, an alkaline material, a cupric salt and a surfaceactive agent capable of forming a substantially monomolecular film atthe liquid-vapor interphase of the fluid being tested.

BACKGROUND OF THE INVENTION The estimation of glucose in urine is atpresent the best practical means available to the physician, as well asto the diabetic patient, for evaluating and controlling the intake ofsugars, effectiveness of antidiabetic medicaments, and the generalcondition of the individual suffering from diabetes mellitus. In orderto be effective, such sugar tests must be simple, rapid, capable ofbeing performed daily by non-technical personnel and must be reasonablyquantitative over a range of pathological urine glucose concentrations.

In addition to clinical testing, there is a need in industry, andparticularly in food and beverage processing industries, for a glucoseor reducing sugar test having the above-noted capabilities. For example,a quantitative determination of sugar in wine can be used as a means forevaluating its dryness. The disappearance or absence of reducing sugarsin beer can be used as a means for determining the progress orcompletion of the fermentation process. Still another example of theneed for a quick and quantitative sugar test can be found in the potatochip processing industry where too much sugar in the raw potatoesresults in a charred or brown chip.

DESCRIPTION OF THE PRIOR ART Methods heretofore available for detectingreducing sugars in urine were based on the principle of heating amixture of measured aliquots of urine and an alkaline copper solution(such as Benedicts, Fehlings, Folin-McElroy) or an alkaline bismuthsolution (such as Nylander, Alment). In qualitative estimations, theprecipitation of cuprous oxide from the cupric compound, or thereduction of the bismuth ion to black metallic bismuth, indicates thepresence of a reducing carbohydrate.

In quantitative urine sugar estimations, the usual method employedheretofore has been that of Benedict (Journal of the American MedicalAssociation, vol. 57, page 1193, 1911). This involves adding measuredamounts of urine to an aliquot of a standard solution, while boiling thelatter, until the blue cupric compound contained therein is completelydecolorized, forming cuprous thiocyanate. By a calculation, thepercentage of reducing substance in the urine is determined.

Sheftel, US. Pat. No. 1,769,862, has described a method for thequantitative determination of sugar in urine based on the followingsteps: (a) Measured aliquots of a special modified Benedict solutioncontaining creatinine and a hydrophilic colloid are mixed with ameasured aliquot of urine; (b) the mixture is heated in boiling waterfor five minutes; (c) if a change in the original blue color appears,the solution is compared with a color scale and the correspondingpercentage of sugar is estimated.

3,63%,958 Patented Dec. 28, 1971 All of the procedures thus farmentioned have one great drawback. They all require an extraneous sourceof heat. This drawback was solved by the method described in US. ReissuePat. No. 23,705 which discloses the use of a tablet test compositioncontaining a cupric salt and acid material and an excess of an alkalinematerial such that when the tablet is added to an aqueous fluidcontaining reducing sugars, the exothermic neutralization reactionbetween the acid and base creates suflicient heat to enable the cupricsalt to be reduced to cuprous oxide. This is, of course, accomplishedwithout recourse to any extraneous source of heat. Moreover, thealkaline material is present in excess of the amount required in theneutralization reaction so that the reduction of the cupric salt mayproceed in an alkaline pH environment.

Over the years it has been found that the use of this tablet test systemin fluids containing certain foam-forming or gelling constituents oftenresults in the following adverse test reaction conditions:

( l) The boiling (heating) time of the fluid may be increased as much asfive times that normally required.

(2) The test reaction mixture may lift the tablet at least partially outof the solution, thereby resulting in insuificient heating of the testmixture.

(3) Color changes may be obscured to the extent that the end point isnot readily visible.

(4) The reaction may proceed in a manner such that proper heatdissipation is not achieved, thereby causing erroneous results.

(5) Certain pathological constituents in the test fluid and particularlyin urine, may be coagulated by the exothermic test reaction and cause anuneven color suspension which makes the reading thereof difficult.

Certain of the above adverse reactions in testing urine for glucose havein the past been eliminated by the addition thereto of materials such ascaprylic alcohol. Such additions have, however, been made as asequential step after the test procedure has commenced, and thematerials suggested have been liquids or solutions which require the useof additional measuring and dispensing equipment, reagent solutions andso forth.

"OBJECTS OF THE INVENTION It is therefore an object of the presentinvention to provide a single entity test composition of the typedescribed herein which provides uniform results regardless of thepresence of constituents other than reducing sugars in the fluid beingtested.

Other more specific objects are the elimination of the adverse testreaction conditions enumerated hereinabove.

SUMMARY OF THE INVENTION It has now been found that an improved singleunit test composition for detecting reducing sugars in aqueous fluidscontaining ingredients which have heretofore caused the aforesaidadverse test reaction conditions is provided by a combination of (a) acupric salt, (b) an acid material, (c) an alkaline material or mixtureof alkaline materials and (d) a surface active agent which tends to forma continuous substantially monomolecular film at the liquid vaporinterphase of the fluid being tested, said surface active agent beinguniformly present in the test composition on a solid, particulatecarrier material therefor.

DESCRIPTION OF THE PREFERRED EMBODIMENT The basis of all test systemscontaining alkaline cupric ions heretofore employed is: (a) a cupricsalt, such as cupric sulfate; (b) an alkaline-reacting substance, suchas sodium carbonate, and/or potassium hydroxide; and, (c) a member ofthe group of salts that are capable of holding cupric hydroxide insolution. Fehling (Annalen, vol. 72, page 106, of 1849) used normalalkali-metal tartrates (Rochelle salts), while enedict (J. Biol. Chem.vol. 3, page 101, of 1907) used normal alkali-metal citrates andFolin-McEllroy (J. Biol. Chem. vol. 33 page 513, of 1918) used normalalkali-metal phosphates.

Stable test compositions of excellent specificity for reducingcarbohydrates, and fulfilling the objects of the present invention, maybe obtained by preparing solid, dry mixtures of the followingingredients in proper proportions:

(a) A water soluble cupric salt;

(b) A solid alkali material of the group consisting of the oxides andhydroxides of the alkali metals;

(c) A member of the group of solid acids and acid reacting salts formingwater-soluble complexes with cupric ions in alkaline solution, whichgroup consists of citric acid, tartaric acid, the alkali metal acidsalts of citric acid; and,

(d) A surface active agent (as will be described hereinafter) and asolid, particulate carrier therefor.

Preferably, for convenience of the user, the dry mixtures are tabletedso that each tablet will be sufficient to make one analyticaldetermination. When these compositions, either in powder or tablet form,are mixed with a small amount of an aqueous specimen such as urine, theydissolve rapidly with the evolution of a considerable amount of heat. Itmay also be desirable to dilute the test solution with water. In theabsence of a reducing carbohydrate, a clear blue solution is obtained.In the presence of a reducing sugar, a green, yellow or brownish redprecipitate of cuprous oxide is obtained, which is held in suspension bythe highly concentrated alkaline solution. To determine quantitativelythe amount of reducing carbohydrate present in the test specimen, thecolor of the suspension may be compared with panels of a standard colorchart for known concentrations of glucose (or other reducingcarbohydrates) in urine (or any other specimen being tested).

In accordance with Beers law, on which the entire art of colorimetry isbased, the intensity of the observed color of the precipitate isdirectly proportional to the concentration of the colored substance(i.e., cuprous oxide). However, since the concentration of the cuprousoxide is also directly proportional to the concentration of reducingcarbohydrates originally present in the specimen, the observed color islikewise directly proportional to the concentration of the reducingsubstance.

The surface active agents of the present test composition are thosesubstances which when intermixed with the t test composition and addedto the fluid being tested tend to form inelastic substantiallymonomolecular films at the liquid-vapor interphase of the fluid beingtested for reducing sugars. Such materials include, inter alia, highmolecular weight diamides or polyamides of substances such as the loweraliphatic and aromatic diamines or polyamines, castor oil, polarvegetable oils, polyethenoxy esters and ethers, polyglycols, highermolecular weight sulfonamides and disulfonamides, imidazolines of highmolecular weight, silicone oils, fatty alcohols of medium molecularweight, lower phosphate esters such as tri butyl phosphate, beeswax,cellulose ethers and microcrystalline wax. It will be appreciated thatcertain of these surface active substances are solids at roomtemperature and in order that they may be use as described hereinbelow,they must be dissolved in a suitable organic solvent system.

It has unexpectedly been found that although such materials can be usedalone as a sequential addition to the test system, in order toincorporate same directly into a test composition, a carrier plus thesurface active agent must be used. Such carriers are solid, inertsubstances upon or in which the surface active agent is uniformlydispersed or admixed. Exemplary of such carriers are, sodium silicate,aluminum silicate, infusorial earth, talcum powder, titanium dioxide andkaolin. The preferable carriers are the silicates, such as sodiumsilicate, magnesium silicate, aluminum silicate and mixtures thereof.

The surface active agents are incorporated with the carrier prior toadmixing same with the test composition, by simply grinding together ina mortar, blending, or other means of admixing ingredients. If thesurface active agent is a liquid or oil, it may be dissolved in asolvent, added to the inert carrier and the solvent evaporated to leavean even dispersal of the surface active agent on or in the carrier.

Referring now to concentrations of surface active agents and the carrierthereof, it has been found that a wide range of concentration may beutilized, depending upon the type and activity of the surface activeconstituent being used. Generally speaking, however, from about .0l% byweight to about 1.0% by weight of surface active agent is used in a testcomposition while the ratio of surface active agent to carrier may beadjusted to give the maximum amount of surface active agent per unitweight of carrier and still have a free flowing powder. Generallyspeaking the ratio of surface active agent to carrier may be from about1:60 to 0.6:1.

If desired, dilutents, excipients and ancillary or auxiliary agents maybe incorporated into the dry compositions. Thus, a minor proportion ofan alkali metal bicarbonate or carbonate (e.g. sodium bicarbonate orcarbonate) may be added. When the dry composition containing such amaterial is added to the liquid specimen being tested, the solid acid oracid reacting salt therein will interact with the alkali metalbicarbonate or carbonate liberating gaseous carbon dioxide. Theelfervescence thus produced serves to accelerate the mechanicaldisintegration of the composition, especially if it is in tablet form.The reaction is thus speeded up, and the determination is effected morepromptly. Since the total heat evolution is thereby effected within ashorter period, there is less loss of heat by radiation, conduction andvaporization, and traces of reducing carbohydrates are detectable, whichmight otherwise go undetected.

A desiccating agent, such as calcium chloride, and diluents andexcipients may be employed without adversely affecting the sensitivityand specificity of the compositions. Auxiliary agents, such ascreatinine and the hydrophilic colloids, may be incorporated, theirpresence in the test solution being advised by Sheftel, to prevent theformation of the red form of cuprous oxide.

Typical examples of ingredients from group (a) above that may be used(i.e water soluble cupric salts) in preparing the compositions arecupric sulphate, cupric chloride, and cupric acetate.

Typical examples of members of group (b) (i.e. oxides and hydroxides ofalkaline metals) that may be used in the diagnostic compositions aresodium hydroxide and potassium hydroxide.

Typical examples of ingredients of group (0) (i.e. solid acids and acidreacting salts which form water soluble complexes with cupric ions inalkaline solution) that may be used in preparing the dry diagnosticcomposition are: citric acid (monohydrate) and, tartaric acid.

Because of the hygroscopicity of compositions within the scope of thisinvention, it is desirable, although by no means imperative, to useanhydrous reagents instead of the corresponding hydrates. Thus anhydrouscupric sulfate yields more stable compositions than the hydrate salt andthis greater stability holds for all of the ingredients having two formsof high and low (or no) water content. However, if a hydrate does nothave a tendency to deliquesce at the temperature encountered in thelocalities where these compositions are to be used, it may be used in apreparation of the present invention.

The heat evolved during the reaction of the test compositions of thisinvention with urine, or similar specimens, is predominantly due to twoexothermic reactions. One of these is the heat of solution of the alkalimetal J oxide, or hydroxide, in the aqueous fluid. The other is the heatof neutralization of the solid acid or acid reacting salt with thealkali metal hydroxide. The heat evolved will suffice to effect thereduction of the cupric complex to cuprous oxide, which forms aprecipitate of characteristic color.

The dry compositions may also be used for the qualitative detection andquantitative estimation of reducing carbohydrates other than glucose.Thus, they may be used to detect pentosuria, to estimate the amount ofgalactose in urine during a galactose tolerance test for hepaticfunction or hyperthyroidism, or the amount of lactose in milk.

As used and claimed herein, the term detecting or detection means boththe qualitative and quantitative estimations of reducing sugar in thefluid being tested.

EXAMPLE 1 Preparation of test reagent composition A basic testcomposition formulation was made to contain the following ingredientsand quantities thereof per test unit:

Mg. Copper sulfate 19.35 Sodium hydroxide 232.5 Citric acid J 200.0Sodium carbonate 80.0

A second mix was then prepared by adding 15.0 ml. of n-octyl alcohol to20.0 grams of sodium silicate in a mortar and grinding with a pestleuntil the alcohol was evenly dispersed in or on the silicate. The resultwas a fine, particulate admixture of surface active agent and carriertherefor. The second mix was then added to and mixed well with the basictest composition in a weight ratio of 1 mg, of second mix per test unit(532 mg.) of basic mix. The admixture was then formed into tablets, eachtablet having a test unit quantity of ingredients. Other admixtures ofn-octyl alcohol-sodium silicate were prepared and added to the basictest composition at a rate of from about 0.25 mg. to about 3 mg. pertest unit. The preferable range for this admixture was found to be 0.3mg. to 1.5 mg. per test unit,

EXAMPLE 2 Example 1 was repeated except that silicone oil (dimethylpolysiloxane having a viscosity equal to about 60,000 centistokes at 25C.) was substituted for the noctyl alcohol. Optimum concentration ofsilicone oilsodium silicate was found to be from about 0.5 mg. to aboutmg. per test unit.

EXAMPLE 3 Example 1 was repeated except that talcum powder wassubstituted for the sodium silicate.

EXAMPLE 4 Comparison of present test compositions to prior art testcompositions Two drops of urine containing 300 mg. added protein (bovineserum albumin) and 1% added glucose and 10 drops of distilled water wereadded to each of two small test tablets labeled No. 1 and 2. Two dropsof the same urine sample but without the added protein and 10 drops ofdistilled water were added to a third test tube labeled No. 3 A tabletprepared as described in Example 1 was added to the No. 1 tube andtablets prepared as in Example l but without the 1 mg. of surface activeagent and carrier were added to each of the tubes numbered 2 and 3. Theboiling times for the tubes numbered 1 and 3 were substantially the same(20 seconds) while the boiling time for tube N0. 2 was fifty-fiveseconds longer seconds). Moreover the tablet in test No. 2 tended tolift out of the tube while the boiling of tube Nos. 1 and 3 was even andcontrolled. Thus, the use of a surface active agent in a test reactionsystem containing a pathological constitutent (protein) resulted in anormal boiling time, whereas when the surface active agent is notpresent, the boiling time is three to four times longer than normal,

What is claimed is:

1. In a test composition in dry solid form for detecting reducing sugarsin aqueous solution, said composition utilizing a mixture of a watersoluble solid cupric salt, a solid acidic material, and a quantity ofsolid alkaline material in excess of that required to neutralize theacidic material in an amount adequate to generate during suchneutralization sufficient heat for the conversion of the cupric salt tocuprous oxide by the action of any reducing sugar present, theimprovement which comprises the inclusion in the test composition of asurface active agent which tends to form an inelastic substantiallymonomolecular film at the liquid-vapor interphase of the fluid beingtested for reducing sugars, said surface active agent being dispersed insaid test composition in admixture with a solid carrier materialtherefor.

2. The test composition of claim 1 wherein the alkaline material is analkali metal hydroxide.

3. The test composition of claim 1 wherein the alkaline material is amixture of an alkali metal hydroxide and an alkali metal carbonate.

4. The test composition of claim 1 wherein the acid material is selectedfrom the group consisting of citric acid and tartaric acid.

5. The test composition of claim 1 wherein the cupric salt is cupricsulfate.

6. The test composition of claim 1 wherein the surface active agent isselected from the groups consisting of high molecular weight diamidesand polyamides, castor oil, polar vegetable oils, polyethenoxy estersand ethers, polyglycols, high molecular weight sulfonamides anddisulfonamides, imidazolines of high molecular weight, silicone oils,fatty alcohols at medium molecular weight, lower phosphate esters,beeswax, cellulose ethers and microcrystalline wax.

7. The test composition of claim 1 wherein the solid carrier is selectedfrom the group consisting of sodium silicate, aluminum silicate,infusorial earth, talcum powder, titanium dioxide and kaolin.

8. The test composition of claim 1 wherein the surface active agent isn-octyl alcohol and the carrier is sodium silicate.

References Cited UNITED STATES PATENTS 9/1939 Fortune 252408 9/1953Kamlet 252-408 US. Cl. X.R.

zgggy UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,630,958 Dated December 28 1971 Inventor) Helen Mae Free and JohnRobert Wisler It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

Column 1 line 43 Seventh word should read browned," rather than "brown"Column 3 line 65 Ninth word should read used rather than .use"

Column 4 line 13 Fifth word should read therefor "rather than "thereof"Column 4 line 24 Third word should read diluents rather than "dilutents"Column 5 line 60 Insert immediately following 300 mg.

Column 5 line 66 Insert immediately following No. 3

Signed and sealed this 27th day of June 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Atteeting Officer Commissionerof Patents

